PROJECT SUMMARY/ABSTRACT The scientific community is increasingly appreciative of the important role the aryl hydrocarbon receptor (AHR) plays in modulating the host-microbiome metabolic axis. Interestingly, there are strong links between the gut microbiota and the development and/or exacerbation of nonalcoholic fatty liver disease (NAFLD), all of which are modulated by activation or repression of the AHR. At present, however, a direct connection between AHR activation, changes in gut microbiota composition, ceramide synthesis, and NAFLD development has not been established. Further the concept that environmental pollutant-mediated disruption and/or modulation of the AHR-gut microbiota metabolic axis leads to the development of NAFLD has not been addressed. Our published and preliminary data with 2,3,7,8-tetrachlorodibenzofuran (TCDF) indicates AHR activation profoundly alters the gut microbiota in a qualitative and quantitative manner, increases hepatic lipogenesis, and significantly alters hepatic ceramide synthesis through direct AHR activity (a proposed driver of NAFLD). These results have led us to the novel central hypothesis: Dietary exposure to potent environmental AHR ligands leads to functionally significant changes in the gut microbiota that exacerbate NAFLD in a time- and Ahr-dependent manner. Through the innovative use of cutting-edge techniques (metagenomics and metabolomics) and unique mouse models (tissue specific knockouts of Ahr, gnotobiotic mice, and fecal transfer experiments), we plan to identify the functional changes imparted to the gut microbiota following dietary exposure to potent AHR ligands such as TCDF and to evaluate the Ahr-dependent role of ceramides in driving NAFLD pathogenesis and progression. This combination of approaches will allow us to accurately assess bacterial phyla dynamics and its interaction with and impact on the host, and whether these changes result in increased susceptibility to NAFLD. We are particularly interested in how the timing (weaning and adulthood) of the exposure influences disease outcome. This study will provide invaluable insight into the effects of exposure on a number of environmentally relevant persistent AHR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and co-planar PCBs and provide valuable endpoints for future studies in human populations.